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Fusion protein TAT (transactivator of transcription)-OCT4 (octamer-binding transcription factor 4), and coding gene and application thereof

A technology of fusion protein and coding gene, which is applied in the direction of hybrid peptide, recombinant DNA technology, microorganism-based methods, etc., and can solve the problem that there are few studies on the function of individual inducers

Active Publication Date: 2014-03-05
BEIJING ZKZKTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few studies on the function of individual inducers in reprogramming

Method used

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  • Fusion protein TAT (transactivator of transcription)-OCT4 (octamer-binding transcription factor 4), and coding gene and application thereof
  • Fusion protein TAT (transactivator of transcription)-OCT4 (octamer-binding transcription factor 4), and coding gene and application thereof
  • Fusion protein TAT (transactivator of transcription)-OCT4 (octamer-binding transcription factor 4), and coding gene and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, TAT transmembrane peptide nuclear transcription factor construction

[0040] The total RNA of PA-1 cells (purchased from ATCC) was extracted using TRIZOL kit (purchased from Invitrogen), digested with DNase I (purchased from Invitrogen), and the above PA-1 cells were digested with reverse transcriptase Super Transcript III (Invitrogen). The total RNA of 1 cell was reverse-transcribed into cDNA, and using the cDNA as a template, the upstream primer 5'-GCGGAGCTCATGGCGGGACACCTGG-3' and the downstream primer 5'-ATAGCGGCCGCTTATCAGTTTGAATGCA-3' of OCT4 were designed respectively, and the upstream primer of NANOG 5'-GAGCTCATGAGTGTGGATCCAGCTT- 3' and downstream primer 5'-GCGGCCGCTCACACGTCTTCAGGTT-3', with Pfx DNA polymerase (purchased from Invitrogen) was used for PCR reaction.

[0041] The above PCR reaction product was recovered and detected by 1.0% agarose gel electrophoresis. As a result, a 1090bp OCT4 amplification product and a 918bp NANOG amplification pr...

Embodiment 2

[0045] Embodiment 2, expression and purification of TAT transmembrane peptide nuclear transcription factor

[0046] The expression vectors PET-TAT-OCT4, PET-TAT-NANOG and the control empty vector pET28a were transformed into the BL21 Rostta-Gami (purchased from Invitrogen) strain of Escherichia coli E. After induction of expression from Sigma Company) for 5 hours, the bacteria were collected by centrifugation, ultrasonically disrupted, and the target protein was purified through a nickel column (purchased from GE Company) using the HIS tag, and finally the OCT4 antibody (purchased from Santa Cruze Company) was used to ) and NANOG antibodies were used to identify the target protein by western blot. The results of expression purification and identification are as follows: figure 1 shown. The amino acid sequence of the fusion protein (TAT-OCT4) expressed by the expression vector PET-TAT-OCT4 is shown in sequence 8 in the sequence listing. The 1-21st position of sequence 8 in t...

Embodiment 3

[0047] Example 3, Cell transfection detection of TAT transmembrane peptide nuclear transcription factor

[0048] We added TAT-NANOG and TAT-OCT4 with a final concentration of 0.2M to the culture medium of HAF cells, and after 2 hours, we used the antibody of HA (purchased from Sigma Company) to detect the translocation of TAT fusion protein by immunofluorescence. Membrane efficiency was tested. The results showed that the TAT fusion protein had passed through the cell membrane and entered the cytoplasm, and part of the protein had been localized in the nucleus ( figure 2 ).

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Abstract

The invention discloses a fusion protein TAT (transactivator of transcription)-OCT4 (octamer-binding transcription factor 4), and a coding gene and application thereof. The fusion protein provided by the invention comprises a TAT transmembrane peptide and a cell reprogramming correlation factor combined with an amino end or carboxyl end of the TAT transmembrane peptide. After the fusion protein-TAT OCT4 is used for treating HAFs (human aortic fibroblasts), the proliferation of the fibroblasts is promoted, and the enhancement of the expression level of the cyclins in the fibroblasts is promoted. The TAT transmembrane peptide transported nuclei transcription factor protein can be used as a cell reprogramming method, and can have important application prospects in cell induction and clinic.

Description

technical field [0001] The invention relates to a fusion protein TAT-OCT4 and its coding gene and application. Background technique [0002] OCT4 and NANOG are two core transcription factors involved in the maintenance of pluripotency and self-renewal of embryonic stem (ES) cells. At the same time, it also plays a key role in the process of inducing the reprogramming of adult cells into induced pluripotent stem cells (iPS cells) in vitro. In the early studies on iPS cell induction, gene modification methods such as virus transfection were mostly used, but this method has great safety risks. At the end of 2009, it was reported that using polyarginine protein transmembrane peptide and induction factors OCT4, SOX2, c-MYC, KLF4 can also successfully induce human and mouse iPS cells. However, there are few studies on the functions of individual inducers in reprogramming. Contents of the invention [0003] The purpose of the present invention is to provide a fusion protein na...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N5/10C12N1/00C12N5/07C12R1/91
Inventor 戴建武曹佳妮陈冰肖志峰
Owner BEIJING ZKZKTECH CO LTD
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